There is a growing interest in using internal ribosome entry site (IRES) elements for cap-independent expression of foreign genes in eukaryotic cells. Although the number of published nucleotide sequences shown to promote cap-independent translation is increasing rapidly, identification of new IRESes published so far was occasional and accidental and there is no distinct methodology of prediction of IRES activity.
Translation initiation of mRNAs in eukaryotic cells is a complex process that involves the concerted interaction of numerous factors (Pain (1996) Eur. J. Biochem. 236, 747-771). For most mRNAs, the first step is the recruitment of ribosomal 40S subunits onto the mRNA at or near the capped 5′ end (FIG. 1). Association of 40S to mRNA is greatly facilitated by the cap-binding protein complex eIF4F. Factor eIF4F is composed of three subunits: the RNA helicase eIF4A, the cap-binding protein eIF4E, and the multi-adaptor protein eIF4G which acts as a scaffold for the proteins in the complex and has binding sites for eIF4E, eIF4a, eIF3, and poly(A) binding protein.
Infection of cells by a variety of RNA viruses results in the selective inhibition of translation of host but not of viral mRNAs. For example, infection of cells with poliovirus, a cytoplasmic RNA virus, results in the modification of several translation initiation factors. Specifically, the proteolysis of both forms of eIF4G, eIF4GI and eIF4GII (Gradi et al., (1998) Proc. Natl. Acad. Sci. USA 95, 11089-11094) by virally encoded proteases results in inhibition of translation of most capped cellular mRNAs. In contrast, the translation of polioviral mRNA, which contains a 450-nt sequence in the viral 5′ noncoding region (5′NCR) that can recruit 40S subunits in the absence of intact eIF4F, is not inhibited. This sequence element was termed an <<internal ribosome entry site>> or <<IRES>> (Jang et al., 1988. J. Virol. 62, 2363-2643). Such IRES elements have been found in picornaviral, flaviviral, pestiviral, retroviral, lentiviral and insect viral RNAs (Table 1) and animal cellular RNAs (Table 2). IRES containing animal mRNAs can presumably recruit 40S ribosomes both via either their capped 5′ ends or their IRES elements that is likely to make possible the translation under conditions where cap-dependent translation is reduced, for example, during viral infection, at the G2/M phase of cell cycle, and under conditions of stress and apoptosis (Johannes et al., (1999) Proc. Natl. Acad. Sci. USA 96, 13118-13123; Cornelis et al., (2000) Molecular Cell 5, 597-605; Pyronnet et al., (2000) Molecular Cell 5, 607-616; Stein et al., 1998. Mol. and Cell. Biol. 18, 3112-3119; Holcik et al., 2000. Oncogene 19, 4174-4177; Stoneley et al., 2000. Mol. and Cell. Biol. 20, 1162-1169). Up to 3% of cellular mRNAs of animal are translated at reduced concentrations of cap binding complex eIF4F (Johannes et al., (1999) Proc. Natl. Acad. Sci. USA 96, 13118-13123).
Over the past few years a reconstituted ribosome binding assay has allowed for the elucidation of the mechanisms by which various IRES elements work (Pestova et al., 1998. Genes Dev. 12, 67-83). Some of these elements act by providing a high-affinity binding site for the RNA binding surface on eIF4G. Others work by binding to eIF3 and/or the 40S subunit (see FIG. 2). Recently, an important role of direct IRES RNA/18S rRNA interaction has been shown. The Gtx IRES contains several nonoverlapping segments having complementarity to 18S rRNA that were shown to mediate internal initiation of translation (Hu et al., 1999. Proc. Natl. Acad. Sci. USA 96, 1339-1344). Within one of these segments, a 9-nt GC-rich sequence CCGGCGGGU which is 100% complementary to 18S rRNA at nucleotides 1132-1124 was identified. It was shown that synthetic IRESes composed of multiple linked copies of this 9-nt IRES module increased internal initiation dramatically in animal cells (Chappel et al., 2000. Proc. Natl. Acad. Sci. USA 97, 1536-1541).
5′ leaders of several plant viral polycistronic genomic RNAs, including members of the potyviral, comoviral families, are responsible for conferring cap-independent translation. Tobamoviruses and potexvirus X IRESes are located in internal parts of the viral genome (Table 2). Tobacco mosaic tobamovirus (TMV) is a positive-stranded RNA plant virus with a monopartite genome 6395 nucleotides (nt) in length (Goelet et al., 1982. Proc. Natl. Acad. Sci. USA 79, 5818-5822). The 5′ proximal ORFs encoding replicative proteins are expressed directly from the genomic RNA, with the smaller (126 kDa) protein produced approximately >10 times the level of the 183 kDa protein which is produced by occasional readthrough of the stop codon for the 126-kDa ORF (Pelham, 1978, Nature 272, 469-471). Although some replication can occur with only the larger protein, both proteins are required for efficient replication (Ishikawa et al., 1986. Nucl. Acid. Res. 14, 8291-8305). The remaining TMV gene products, the movement protein (MP) and the coat protein (CP), are expressed from 3′ coterminal subgenomic mRNAs (sgRNAs) (reviewed by Palukaitis and Zaitlin, 1986 In: “The plant virus”. M. H. V. van Regenmortel and M. Fraenkel-Conrat, Eds. Vol. 2, pp. 105-131. Plenum Press, NY). Thus, the internal movement protein (MP) gene and the 3′-proximal coat protein gene cannot be translated from genomic RNA of typical tobamoviruses (TMV UI is the type member of the genus Tobamovirus). The dicistronic intermediate-length RNA-2 called sgRNA I2 RNA is translated to produce the 30-kDa MP (Bruening et al., 1976 Virology 71, 498-517; Higgins et al., 1976 Virology 71, 486-497; Beachy and Zaitlin, 1977 Virology 81, 160-169; Goelet and Karn, 1982 J. Mol. Biol. 154, 541-550), whereas the 3′-proximal coat protein (CP) gene of I2 RNA is translationally silent. This gene is expressed only from small monocistronic sgRNA (Siegel et al., 1976 Virology 73, 363-371; Beachy and Zaitlin, 1977 Virology 81, 160-169).
It has been shown (Ivanov et al., 1997, Virology 232, 32-43) that, unlike RNA of typical tobamoviruses, the translation of the CP gene of a crucifer-infecting tobamovirus (crTMV) occurred in vitro by an internal ribosome entry mechanism. The genome of crTMV (6312 nts) contains four traditional genes encoding two components of the replicase (the proteins of 122-kDa and 178-kDa, the readthrough product of 122-kDa), 30-kDa MP and 17-kDa CP (Dorokhov et al., 1993 Dokl. Russian Acad Sci. 332, 518-52; Dorokhov et al., 1994 FEBS Lett. 350, 5-8). It was found that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRESCP148), promoting internal translation initiation of the CP gene and of different reporter genes (Ivanov et al., (1997) Virology 232, 32-43). By analogy with crTMV, the 3′-proximal CP gene of potato virus X occurs by a mechanism of internal initiation (Hefferon et al., 1997 J. Gen. Virol. 78, 3051-3059; Hefferon et al., 2000. Arch. Virol. 145, 945-956). The capacity of crTMV IRESCRCP for mediating internal translation distinguishes this tobamovirus from well-known type member of the genus, TMV U1. The equivalent 148-nt sequence from TMV U1 RNA was incapable (UICP,148SP) of mediating internal translation (Ivanov et al., (1997) Virology 232, 32-43).
Recently, it has been shown that the 228- and 75-nt regions upstream of the MP gene of crTMV and TMV U1 RNAs contained IRES elements, IRESMP,75CR or IRESMP,228CR, which directed expression of the 3′-proximal reporter genes from dicistronic constructs in cell-free translation systems and in isolated protoplasts (Skulachev et al., 1999, Virology 263, 139-154). Moreover, the equivalent sequence from TMV U1 RNA used as the intercistronic spacer (IRESMP,75U1) was able to mediate internal translation of the second gene in dicistronic transcripts.
There are several inventions wherein an IRES element was used for cap-independent expression of foreign gene(s) in linear multicistronic mRNAs via IRES elements in mammalian cells (U.S. Pat. No. 6,060,273; U.S. Pat. No. 6,114,146; U.S. Pat. No. 5,358,856; U.S. Pat. No. 6,096,505; U.S. Pat. No. 171,821; U.S. Pat. No. 5,766,903), plant cells (WO98/54342) and generally, in eukaryotic cells (U.S. Pat. No. 171,821; U.S. Pat. No. 5,766,903; U.S. Pat. No. 5,925,565; U.S. Pat. No. 6,114,146). To provide cap-independent IRES-mediated expression of a gene, a circular RNA was developed as well (U.S. Pat. No. 5,766,903). Cap-independent translation of eukaryotic mRNA could be reached by using 5′UTR of barley yellow dwarf virus RNA that is principally different from known IRESes (U.S. Pat. No. 5,910,628). Generally, all inventions used natural IRESes isolated from animal (e.g. U.S. Pat. No. 5,358,856) or plant viruses (WO98/54342) not having cross-kingdom activity, i.e. these IRESes are limited to either plant or animal cells. There are no inventions developing approaches for the creation of non-natural, artificial IRESes that are capable to provide efficient cap-independent gene expression in animal and plant cells. Moreover, there are no approaches for searching new IRES elements having cross-kingdom activity.
In contrast to animal cell mRNA, there are no published reports on IRES-mediated mRNA translation initiation in plant cells (see Table 2), except for one recent patent application (WO01/59138) describing a plant IRES element of the Arabidopsis RPS18C gene. However, neither the methodology used in this patent application nor the experimental approaches nor the interpretation of the results allows a direct and unambiguous conclusion of an IRES activity in vivo, nor a reliable detection of said elements in plant transcripts. First of all, there was an incomprehensive test for IRES activity. Usually, for detecting IRES activity, the dicistronic mRNA assay is utilised (Pelletier & Sonenberg, 1988, Nature, 334, 320-325). In this test, two types of capped bicistronic mRNAs separated by a putative IRES element (with and without hairpin structure in front of the first cistron) are analysed for the ability to provide expression in vitro and in vivo. The construct without hairpin structure allows ribosome scanning and translation of the first, 5′ proximal cistron, whereas cap-dependent translation of the first cistron is blocked in the construct with hairpin structure. The authors of WO01/59138 tested the putative IRES element of RPS18C only in the artificial bicistronic transcript without hairpin structure. Thus, the results presented in WO01/59138 may well be a consequence of reinitiation of translation (Kozak, 2001, Mol. Cell. Biol 21, 1899-1907). Additionally, it is known that a nucleotide sequence having IRES activity in vitro, frequently does not show IRES activity in eukaryotic cells. WO01/59138 does not contain experimental evidence showing directly and unambiguously that the putative IRES element of Arabidopsis RPS18C is functional in eukaryotic cells (plant or animal).
In contrast to animal cell mRNA, there are no published reports of in vivo cellular mRNA IRES mediated translation initiation in plant cells (Table 2). The low activity of animal virus IRESes (encephalomyocarditis virus IRES, IRESEMCV) in plants was reported (Urwin et al., 2000 Plant J. 24, 583-589). There is no evidence of cross-kingdom (plant, animal, yeast) activity of any IRES element so far. Although the number of published nucleotide sequences that are capable to provide cap-independent translation increases constantly, identification of new IRESes happens only accidentally, and there is no distinct methodology of prediction.
It is therefore an object of the invention to provide a method for identifying novel eukaryotic IRES elements.
It is a further object of the invention to provide novel eukaryotic IRES elements, notably of plant origin.
It is a further object to provide novel IRES elements having cross-kingdom activity.
It is another object of the invention to provide a process of expressing a nucleotide sequence of interest in eukaryotic cell(s) under translational control of a novel IRES element, notably of an IRES element of plant origin.